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Title: Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

Abstract

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs. HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use bymore » binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Authors:
 [1];  [1];  [2];  [2];  [3]
  1. Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.
  2. Emory Univ., Atlanta, GA (United States). Dept. of Pathology and Lab. Medicine
  3. Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab., Dept. of Plant Biology, Plant Resilience Inst., and Howard Hughes Medical Inst.
Publication Date:
Research Org.:
Michigan State Univ., East Lansing, MI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1368384
Grant/Contract Number:
FG02-91ER20021
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Plant Methods
Additional Journal Information:
Journal Volume: 13; Journal Issue: 1; Journal ID: ISSN 1746-4811
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; Protein targeting; Leucine-rich repeat; Variable lymphocyte receptor; Modules HopM1

Citation Formats

Velásquez, André C., Nomura, Kinya, Cooper, Max D., Herrin, Brantley R., and He, Sheng Yang. Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins. United States: N. p., 2017. Web. doi:10.1186/s13007-017-0180-8.
Velásquez, André C., Nomura, Kinya, Cooper, Max D., Herrin, Brantley R., & He, Sheng Yang. Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins. United States. doi:10.1186/s13007-017-0180-8.
Velásquez, André C., Nomura, Kinya, Cooper, Max D., Herrin, Brantley R., and He, Sheng Yang. Wed . "Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins". United States. doi:10.1186/s13007-017-0180-8. https://www.osti.gov/servlets/purl/1368384.
@article{osti_1368384,
title = {Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins},
author = {Velásquez, André C. and Nomura, Kinya and Cooper, Max D. and Herrin, Brantley R. and He, Sheng Yang},
abstractNote = {The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs. HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.},
doi = {10.1186/s13007-017-0180-8},
journal = {Plant Methods},
number = 1,
volume = 13,
place = {United States},
year = {Wed Apr 19 00:00:00 EDT 2017},
month = {Wed Apr 19 00:00:00 EDT 2017}
}

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  • Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less
  • Plant disease resistance genes function in highly specific pathogen recognition pathways. RPS2 is a resistance gene of Arabidposis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.
  • The primary structure of the {beta} chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A {lambda} phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the {beta} chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5{prime} untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding amore » signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3{prime} noncoding region of 307 nucleotides. The 3{prime} noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact {beta} chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the {beta} chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the {alpha} chain of GPIb and nine tandem repeats in leucine-rich {alpha}{sub 2}-glycoprotein. The amino-terminal region of the {beta} chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein.« less
  • Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit themore » level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.« less